Alcohol consumption causes damage to multiple organs, including the liver and brain, and ethanol can affect the epigenetic state of cells by modifying DNA methylation and modifying histones. These epigenetic changes may compromise regeneration of tissues, predispose cells to become neoplastically transformed, and/or alter the function of stem cells or differentiated cells. In pilot experiments testing the effects of ethanol on murine embryonic stem (ES) cells, we observed some striking effects of ethanol and acetaldehyde, a metabolite of ethanol, on both gene expression and on histone modifications. Indeed, we already have data suggesting that alcohol influences the activity of the Polycomb Repressive Complex 2, a multi-protein complex that is a central negative epigenetic regulator of stem cell differentiation. Based on these preliminary results, our hypothesis is that alcohol and acetaldehyde induce epigenetic changes in stem cells, influencing the ability of such stem cells to undergo normal differentiation. In Aim (1) we propose to determine the global effects of alcohol and acetaldehyde on the epigenetic state of murine embryonic stem (ES) cells by performing genome-wide analysis using chromatin immunoprecipitation-sequencing (ChIP-Seq) and quantifying genome-wide changes in DNA methylation by Reduced-Representation Bi-sulfite Sequencing (RRBS). In Aim (2) we will determine how changes in the epigenetic state induced by alcohol and acetaldehyde affect gene expression and the differentiation of ES cells by performing genome-wide analysis using RNA-seq technology and by interpreting these changes in light of the changes in chromatin structure (Aim 1). We will use all-trans retinoic acid (RA), a signaling molecule that induces ES cell differentiation, as a positive control in Aim (1) since we and others have shown that RA can change the epigenetic state of ES cells. Since alcohol causes tissue injury, requiring stem cells in adult tissues to proliferate and differentiate repeatedly in an attempt to repair the damage, ou studies will lead to insights into the mechanisms by which alcohol both causes tissue injury and compromises response to tissue injury. We hypothesize that alcohol-induced epigenetic changes may underlie disorders as diverse as addictive behavior, tumor formation, and cirrhosis of the liver. Moreover, unlike genetic mutations that change the DNA sequence and are generally permanent, epigenetic changes can be reversed and therefore are reasonable targets for therapies to treat alcohol-induced tissue injury.